Detection of bacteria and fungi

ABSTRACT

A method of detecting a ligase expressing micro-organism in a sample comprises steps of treating the sample under conditions that inhibit the activity of ATP-dependent ligase from mammalian cells but which do not inhibit the activity of the microbial ligases, contacting the sample or a portion of the sample with a nucleic acid molecule which acts as a substrate for ligase activity in the sample, incubating the thus contacted sample under conditions suitable for ligase activity; and specifically determining the presence and/or the amount of a ligated nucleic acid molecule resulting from the action of the ligase on the substrate nucleic acid molecule to indicate the presence of the ligase expressing micro-organism. The micro-organism may be a fungus or a bacterium or both. High pH conditions may be employed to inactivate mammalian ligases. Related kits are described.

FIELD OF THE INVENTION

The present invention relates to the field of detecting micro-organisms,in particular distinguishing between infection by bacteria or fungi andyeasts. The methods of the invention are highly sensitive and havenumerous applications. Methods and kits are described which rely uponnovel indicators of microorganism viability.

BACKGROUND TO THE INVENTION

There is a need for rapid detection of bacteria and fungi in clinicalspecimens and there is a requirement to distinguish between a bacterialand fungal infection. Culture approaches can be used but such techniquesrequire several days to complete, especially when attempting to detectsmall numbers of bacteria and also when detecting slower growing microorganisms.

Tests may be carried out based upon measuring the presence of a moleculewhich can be linked to the presence in the sample of a bacterial orfungal cell. The most commonly detected molecule is AdenosineTriphosphate (ATP). Detection of DNA and RNA has also been proposed,although the correlation between the presence of DNA and RNA andviability is not clear-cut due to the variable persistence of nucleicacids in specimens post death of the microorganism (Keer & Birch,Journal of Microbiological Methods 53 (2003) 175-183). Detection ofadenylate kinase as an indicator of viability has also been proposed(Squirrel) D J, Murphy M J, Leslie R L, Green J C D: A comparison of ATPand adenylate kinase as bacterial cell markers: correlation with agarplate counts. In Bioluminescence and chemiluminescence progress andcurrent applications. Edited by: Stanley R A, Kricka L J. John Wiley andSons; 2002 and WO 96/02665)

A routinely employed method for determining ATP levels involves the useof bioluminescence. The method uses the ATP dependency of the reactionin which light emitting luciferase catalyzes oxidation of luciferin. Themethod may be used to measure relatively low concentrations of ATP. Kitsuseful for detecting ATP using bioluminescence are commerciallyavailable from Roche, New Horizons Diagnostics Corp, Celsis etc.

Ligases are enzymes which catalyze ligation of nucleic acid molecules.The ligation reaction requires either ATP or NAD+ as co-factor dependingupon the ligase concerned. WO 2009/007719 describes the use of NADligases to detect viable bacteria.

SUMMARY OF THE INVENTION

The present invention describes a procedure for detecting ligaseexpressing microorganisms, such as fungi (and bacteria) in a sample,such as a mammalian specimen or sample containing mammalian cells, bymeasuring the (ATP-dependent and/or NAD-dependent) ligase present in thesample after lysis of any fungal cells present, typically following abackground reduction step to remove mammalian ATP-dependent ligaseactivity. The invention also describes a procedure for distinguishingbetween fungal and bacterial cells by measuring both the NAD-dependentligase content in a sample and the ATP-dependent ligase content. If onlyATP-dependent ligase is present then only fungal cells are present, ifboth enzyme activities are present then a mixed population ofbacterial/fungal cells are present. If only NAD-dependent ligaseactivity is present the sample contains bacterial cells only.

In a first aspect, the invention provides a method of detecting a ligaseexpressing micro-organism in a sample comprising:

(a) treating the sample under conditions that inhibit the activity ofATP-dependent ligase from mammalian cells but which do not inhibit theactivity of the microbial ligases,(b) contacting the sample or a portion of the sample with a nucleic acidmolecule which acts as a substrate for ligase activity in the sample,(c) incubating the thus contacted sample under conditions suitable forligase activity; and(d) specifically determining the presence and/or the amount of a ligatednucleic acid molecule resulting from the action of the ligase on thesubstrate nucleic acid molecule to indicate the presence of the ligaseexpressing micro-organism.

In certain embodiments, the ligase expressing micro-organism comprisesfungal or bacterial cells or both. The ligase expressed by themicro-organism may thus comprise an ATP-dependent ligase, anNAD-dependent ligase or both depending upon the cell types present inthe sample. The presence of NAD-dependent ligase activity in the sampleindicates the presence of bacterial cells (in particular eubacterialcells). Employment of suitable conditions in the sample, or a portionthereof, may permit NAD and ATP-dependent ligase activity to bedetermined respectively, as discussed herein.

The invention also provides a method of detecting an ATP-dependentligase expressing micro-organism in a sample comprising:

(a) contacting the sample or a portion of the sample with a nucleic acidmolecule which acts as a substrate for ATP-dependent ligase activity inthe sample,(b) incubating the thus contacted sample under conditions suitable forATP-dependent ligase activity; and(c) specifically determining the presence and/or the amount of a ligatednucleic acid molecule resulting from the action of the ATP-dependentligase on the substrate nucleic acid molecule to indicate the presenceof the ATP-dependent ligase expressing micro-organism.

In specific embodiments, the ATP-dependent ligase expressingmicro-organism comprises fungal or bacterial cells or both. In preferredembodiments, the methods further comprise, prior to step

(a), treating the sample under conditions that inhibit the activity ofATP-dependent ligase from mammalian cells but which do not inhibit theactivity of the microbial ATP-dependent ligases. Suitable conditions arediscussed in detail herein. These methods may additionally comprise:(d) contacting the sample or a portion of the sample with a nucleic acidmolecule which acts as a substrate for NAD-dependent ligase activity inthe sample,(e) incubating the thus contacted sample under conditions suitable forNAD-dependent ligase activity; and(f) specifically determining the presence (and/or the amount) of aligated nucleic acid molecule resulting from the action of theNAD-dependent ligase on the substrate nucleic acid molecule to indicatethe presence of bacterial cells in the sample.

In such methods, the presence of ATP-dependent ligase activity andabsence of NAD-dependent ligase activity in the sample indicates thatthe sample contains fungal cells or a non-bacterial micro-organism. Incertain embodiments, for example where no discrimination is neededbetween bacterial or fungal cells in the sample, the same nucleic acidmolecule may be used as a substrate for both NAD-dependent ligaseactivity and ATP-dependent ligase activity.

Accordingly, the invention provides a method of detecting fungal orbacterial cells or both comprising:

(a) contacting the sample with a nucleic acid molecule which acts as asubstrate for ATP-dependent ligase activity in the sample,(b) incubating the thus contacted sample under conditions suitable forATP-dependent ligase activity; and(c) specifically determining the presence (and/or the amount) of aligated nucleic acid molecule resulting from the action of theATP-dependent ligase on the substrate nucleic acid molecule to indicatethe presence of fungi and/or bacteria(d) contacting the sample with a nucleic acid molecule which acts as asubstrate for NAD-dependent ligase activity in the sample, which may bethe same nucleic acid molecule as is used as the substrate for ATPligase(e) incubating the thus contacted sample under conditions suitable forNAD-dependent ligase activity; and(f) specifically determining the presence (and/or the amount) of aligated nucleic acid molecule resulting from the action of theNAD-dependent ligase on the substrate nucleic acid molecule to indicatethe presence of bacteria only

The invention also provides a method of distinguishing fungal cells frombacterial cells present in a sample with inhibition of the mammalianbackground from ATP-dependent ligase prior to lysis of the fungal andbacterial cells and detecting the released fungal ATP-dependent ligaseor the released bacterial NAD-dependent ligase.

Accordingly, in a further aspect the invention provides a method ofdetecting fungal or bacterial cells or both comprising:

(a) treating the sample under conditions that inhibit the mammalianbackground signal from ATP-dependent ligase but which do not affectfungal ATP and microbial NAD-dependent ligases(b) lysing the sample to release the fungal ATP and bacterialNAD-dependent ligases(c) contacting the sample with a nucleic acid molecule which acts as asubstrate for ATP-dependent ligase activity in the sample,(d) incubating the thus contacted sample under conditions suitable forATP-dependent ligase activity; and(e) specifically determining the presence (and/or the amount) of aligated nucleic acid molecule resulting from the action of theATP-dependent ligase on the substrate nucleic acid molecule to indicatethe presence of fungi and/or bacteria(f) contacting the sample with a nucleic acid molecule which acts as asubstrate for NAD-dependent ligase activity in the sample,(g) incubating the thus contacted sample under conditions suitable forNAD-dependent ligase activity; and(h) specifically determining the presence (and/or the amount) of aligated nucleic acid molecule resulting from the action of theNAD-dependent ligase on the substrate nucleic acid molecule to indicatethe presence of bacteria only

A “sample” in the context of the present invention is defined to includeany sample in which it is desirable to test for the presence of eitherbacteria expressing an NAD-dependent ligase or fungi expressing anATP-dependent ligase or both. Thus, the sample is generally a samplesuspected of containing, or in some circumstances known to contain, amicro-organism. Detection of ligase activity in the sample is consideredindicative of the presence of the micro-organism. The sample isgenerally one that may contain mammalian cells, which also expressligase activity. However, the methods of the invention permit ligaseactivity in the sample resulting from the presence of mammalian cells tobe selectively removed (through inactivation) prior to detection ofmicrobial ligase activity. The step of removing any mammalian cellATP-dependent ligase activity may not be necessary in samples where itis known that no mammalian cells are present. This may be the case forexample where NAD versus ATP dependent ligase activity is investigatedto determine whether fungal and/or bacterial cells are present in thesample.

Thus the sample may comprise, consist essentially of or consist of aclinical sample, or an in vitro assay system for example. Samples maycomprise, consist essentially of or consist of beverage or food samplesor preparations thereof, or pharmaceutical or cosmetic products such aspersonal care products including shampoos, conditioners, moisturisersetc., all of which are tested for microbial contamination as a matter ofroutine. The sample may comprise, consist essentially of or consist oftissue or cells and may comprise, consist essentially of or consist of asputum or a blood sample or a platelet sample for example.

By “ATP-dependent ligase” is meant an ATP ligase which depends upon theadenosine triphosphate (ATP) cofactor for activity. The activity of theATP-dependent ligase is the formation of a phosphodiester bond betweenthe 5′ end of a nucleic acid molecule and the 3′ end of a nucleic acidmolecule. By “NAD-dependent ligase” is meant a DNA ligase which dependsupon the nicotinamide adenine dinucleotide (NAD+) cofactor for activity.NAD-dependent ligases can be distinguished from ATP-dependent ligaseswhich rely upon the cofactor ATP for activity. The activity of theNAD-dependent ligase is the formation of a phosphodiester bond betweenthe 5′ end of a nucleic acid molecule and the 3′ end of a nucleic acidmolecule.

The methods of the present invention provide significant technicaladvantages, due in large part to the fact that a novel nucleic acidmolecule is generated as part of the method. In the methods of thepresent invention, unreacted nucleic acid molecule will not contributeto the signal, and as a result no false positive signals should beproduced when the methods are carried out.

Furthermore, the method is highly sensitive providing detection of theATP and NAD-dependent ligases present in the sample down to femtogramand possibly even attogram levels. The sensitivity is derived from thefact that every bacterial or fungal cell contains thousands of enzymemolecules, and thus each can catalyse multiple ligation events undersuitable conditions. Every bacterial and fungal cell must produce ligaseactivity to repair ongoing genomic damage and this essential activitycontributes to its usefulness as a marker for the presence of viablemicrobial cells. Thus unlike PCR approaches, which must target one or afew copies of a gene per cell or use additional steps or reagents todetect ribosomal or messenger RNA, the approach described herein targetsthe detection of multiple copies of the ATP and NAD-dependent ligase percell in a simple assay format. The sensitivity is further enhancedcompared to other approaches in that each copy of the ligase is able tomodify multiple (hundreds or thousands) substrate nucleic acid moleculeswhich can each then be detected.

Depending on the sample type which may contain host ATP dependentligases it may be beneficial to inactivate these host ligases bypretreating the sample in such a way that the host ligases areinactivated but the fungal or bacterial ligases remain active. Theapproach described herein may, in certain embodiments, rely on thedifference in structure of mammalian cells and bacterial or fungalcells. Conditions are described that lyse or solubilise the mammaliancell membrane but courtesy of the fungal and bacterial cell walls, leavethe fungal and bacterial membranes intact. Once the ligases are releasedfrom the mammalian cells they are exposed to conditions that furtherinactivate these released ligases. Conditions used for lysis of themammalian membranes include the use of detergents that may or may not beused in conjunction with high or low pH. Conditions used for theinactivation of released ligases include the use of high or low pH.

Thus, in certain embodiments the methods of the invention comprisetreatment of the sample with an agent that permeabilizes the cellmembrane of mammalian cells in the sample. The agent preferably does notsignificantly permeate the cell wall of any micro-organisms in thesample. The agent may be a detergent, many suitable examples of whichare known in the art and commercially available. One specific exampleshown to be effective herein is the surfactant/detergent Triton X-100.

As indicated above, the conditions that inhibit the activity ofATP-dependent ligase from mammalian cells but which do not inhibit theactivity of the microbial ligases may comprise high or low pH. High pHis generally a pH of at least around 10, such as around 10, 11, 12, 13or 14. Low pH is generally a pH of less than or equal to around 4, suchas around 4, 3, 2, or 1. Altering the pH of the sample may be achievedusing any suitable means, as would be readily appreciated by one skilledin the art. It is shown herein that microbial ligases are surprisinglyresistant to extremes of pH, whereas mammalian ligases are inactivatedunder the same pH conditions. This permits selective detection ofmicrobial ligases in a sample containing both mammalian cells andmicrobial cells. In specific embodiments, the conditions that inhibitthe activity of ATP-dependent ligase from mammalian cells but which donot inhibit the activity of the microbial ligases comprise treating thesample with sodium hydroxide (NaOH) or sodium carbonate (Na2CO3). Suchagents can readily be used, as shown herein, to increase the pH of thesample to high pH thus inactivating mammalian ligase activity whilstleaving the microbial (fungal and bacterial) ligases active. Suitableconcentrations and volumes of the appropriate agent can be applied by askilled person. In certain embodiments, however, the NaOH is around 5 mMNaOH. In further embodiments, the pH is around 12 to inactivatemammalian ATP-dependent ligase activity (but not microbial ligases). Inyet further embodiments, the treatment are carried out for around 20minutes. Suitable agents for lowering the pH to less than or equal toaround 4 include acids such as hydrochloric acid (HCl) and sulphuricacid (H2SO4).

In specific embodiments, pH conditions may be increased to at leastaround 11, or at least around 11.2. This treatment may result in lysisof micro-organisms in the sample and thus lead to ligase release intothe sample. This permits detection of ligases in the sample, originatingfrom the micro-organism, without the need for a separate cell lysisstep. Under these conditions, mammalian ligases (such as bloodATP-dependent ligases) are inactivated.

In other embodiments, the methods of the invention further compriselysis of micro-organisms (fungi or bacteria) in the sample to releaseATP and NAD-dependent ligase. This step is preferably carried out beforethe sample is contacted with the nucleic acid substrate, although thisis not essential. Thus, the methods of the invention may furthercomprise, following the treatment step, lysing the sample to release themicrobial ligase. However, as shown herein, microbial ligases are muchmore resistant to high pH conditions than mammalian ligases. Thus, themethods of the invention may incorporate a lysis step to lyse all cellsin the sample, irrespective of their origin (i.e. to include bothmicro-organisms and mammalian cells). Following this lysis step, themammalian ligases can be selectively inactivated, for example using highor low pH conditions, and the ligases expressed by any micro-organismsin the sample detected according to the methods of the invention.

In specific embodiments, lysis is performed mechanically, although lysismay also be performed chemically. Suitable agents for lysing bacterialand fungal cells selectively are known in the art and include bacterialprotein extraction reagents such as B-PER (Pierce) and Y-PER (Pierce)for example. Mechanical be achieved through sonication or French Pressor ribolysis (‘bead beating’) for example. However, lysis may not beessential in all embodiments of the invention. In particular, increasingthe permeability of the bacterial or fungal cell wall and/or membranemay in certain embodiments be sufficient to enable detection of ATP orNAD-dependent ligase activity according to the methods of the invention.Suitable agents and techniques for achieving this increase inpermeability are known in the art and include high pH conditions asdescribed herein.

As stated herein, one step in the ligase assay methods of the inventioncomprises, consists essentially of or consists of contacting the samplewith a nucleic acid molecule which acts as a substrate for microbial(ATP or NAD-dependent) ligase activity in the sample. Any suitableligatable molecule which can be specifically detected once ligated maybe utilised in the methods of the invention.

For the avoidance of doubt, it is hereby stated that the ligated nucleicacid molecule is generally a novel detectable nucleic acid moleculewhich has a different overall structure to that of the original(substrate) nucleic acid molecule. Thus, the novel detectable nucleicacid molecule may contain additional nucleotides such that the novelnucleic acid molecule may be uniquely identified, for example byamplification utilising primers which can only bind and produce anamplification product using the ligated nucleic acid molecule as atemplate. However, it may be that only one strand is extended ascompared to the (original) substrate nucleic acid molecule, for examplethe ligase may seal a nick in one strand of a double stranded substratemolecule.

The substrate nucleic acid molecules for use in the methods, andinclusion in the kits, of the invention, must be of sequence andstructure such that the ATP or NAD-dependent ligase can act on themolecule as the case may be to produce detectable ligated (novel)nucleic acid molecule.

Suitable substrate nucleic acid molecules for use in the inventioncomprise, consist essentially of or consist of the nucleotide sequencesset forth as SEQ ID NO: 1, 2 and 3 and SEQ ID NO: 6, 7, and 8respectively and described in more detail in the experimental sectionbelow. It is noted that variants of these sequences may be utilised inthe present invention. For example, additional flanking sequences may beadded. Variant sequences may have at least 90%, at least 91%, at least92%, at least 93%, at least 94%, at least 95%, at least 96%, at least97%, at least 98%, or at least 99% nucleotide sequence identity with thenucleotide sequences of the substrate nucleic acid. The nucleic acidmolecules may incorporate synthetic nucleotide analogues as appropriateor may be RNA or PNA based for example, or mixtures thereof. They may belabelled, such as using a fluorescent label, or FRET pair, in certainembodiments to facilitate detection. Suitable detection methods aredescribed herein.

“Nucleic acid” is defined herein to include any natural nucleic acid andnatural or synthetic analogues that are capable of being ligated by anATP or NAD-dependent ligase in order to generate a ligated (noveldetectable) nucleic acid molecule. The ligation reaction may involveeither joining of two DNA molecules or sealing a nick in a nucleic acidmolecule to produce a detectable ligated nucleic acid molecule forexample. Suitable nucleic acid molecules may be composed of, forexample, double or single-stranded DNA and double or single-strandedRNA. Nucleic acid molecules which are partially double-stranded andpartially single-stranded are also contemplated in certain embodimentsof the invention. In certain embodiments the substrate nucleic acidmolecule comprises, consists essentially of or consists of dsDNA, toinclude nicked dsDNA. The term “nucleic acid” encompasses syntheticanalogues which are capable of being ligated by ATP or NAD-dependentligase in a sample in an analogous manner to natural nucleic acids, forexample nucleic acid analogues incorporating non-natural or derivatizedbases, or nucleic acid analogues having a modified backbone. Inparticular, the term “double-stranded DNA” or “dsDNA” is to beinterpreted as encompassing dsDNA containing non-natural bases.

Though the nucleic acid substrate may comprise, consist essentially ofor consist of a blunt-ended double-stranded DNA molecule, in a separateembodiment the nucleic acid substrate for the ATP or NAD-dependentligase comprises, consists essentially of or consists of two doublestranded DNA molecules with a complementary overhang and 5′ phosphategroups at the ends to be joined. In one specific embodiment, thecomplementary overhang is between 2 and 10, such as 3 or 5 base pairs.In an alternative embodiment, the nucleic acid substrate comprises,consists essentially of or consists of a partially double-stranded DNAmolecule with a nick containing a 5′ phosphate. Synthesized nucleic acidmolecules are commercially available and can be made to order with aterminal 5′ phosphate group attached. This has the technical advantagethat 100% of the nucleic acid molecules used in the methods of theinvention will be labelled with a 5′ phosphate group. Furthermore, thenucleic acid substrates can be designed to specification, for example toinclude biotin molecules for subsequent post-ligation capture if sodesired, as described herein.

Thus, in embodiments of the invention, the novel nucleic acid moleculethat is detected is generated by ligation of the 3′ end of the nucleicacid molecule to the 5′ end of a further nucleic acid molecule. In theseembodiments, if the ligase is present in the sample, it will catalysethe ligation and a ligated nucleic acid molecule (incorporating anoverall novel sequence) will be formed which can be detected by asubsequent process, as detailed herein (such as a nucleic acidamplification process for example).

Thus, the substrate nucleic acid molecule may, in fact, comprise,consist essentially of or consist of two or more nucleic acid moleculesas appropriate. This applies generally to the methods and kits of theinvention.

In certain embodiments, the nucleic acid substrate comprises, consistsessentially of or consists of two double stranded nucleic acid moleculeswith single-stranded complementary overhangs.

The 3′ end of nucleic acid substrate molecules that are not productivelyjoined in terms of producing a ligated product which is then detected(desired to be joined) may be blocked with a suitable blocking group inorder to ensure that they cannot participate in a ligation reaction. Anyappropriate blocking group may be utilised.

In specific embodiments, the nucleic acid molecule which acts as asubstrate for ATP or NAD-dependent ligase activity in the samplecomprises, consists essentially of or consists of a nicked doublestranded nucleic acid molecule. In specific embodiments, the overallsubstrate may be made up of three specific single stranded DNA (ssDNA)molecules. Two or more of the ssDNA molecules may be of identicalsequence. One ssDNA molecule may hybridize to the other two nucleic acidmolecules in a manner such that a double stranded region is formed thatcontains a nick. NAD-dependent ligase activity, if present in thesample, may seal the nick thus producing a double stranded DNA moleculewhich can be detected according to the methods described herein.

In further specific embodiments, the nucleic acid molecule which acts asa substrate for ATP or NAD-dependent ligase activity in the samplecomprises, consists essentially of or consists of two nucleic acidmolecules which can be ligated together.

Preferably, the nucleic acid substrate is present in excess, and inparticular in large molar excess, over the ligase in the sample. This isan important technical distinction over prior art methods. Because anovel ligated nucleic acid molecule is detected, only the presence ofthis molecule in the sample is essential for the detection methods towork effectively. Thus, it is not detrimental to the methods of theinvention if other nucleic acid molecules are present in the sample suchas from the bacteria or fungi to be detected or from mammalian sourceswhich may be found in the sample to be tested for example.

Preferably, the substrate nucleic acid molecules are designed such thatthey do not have high levels of homology with the genome of the one ormore bacteria or other micro-organisms which produce the ATP orNAD-dependent ligase which is to be detected in the sample. This meansthat, even in the presence of contaminating nucleic acid molecules, onlythe novel ligated nucleic acid molecule may be detected. Thus, thesubstrate should have sufficiently low levels of sequence identity withthe genomic DNA of the bacteria or fungi to be detected to preventnon-specific amplification of genomic DNA producing a false positiveresult. The sequence of the substrate may thus be designed with thetarget bacteria in mind. In particular, the primers for amplifyingspecifically the novel ligated nucleic acid molecule are designed suchthat they do not produce an amplification product from the bacterialgenomic DNA. For example, the substrate and primers may incorporatecomplementary non-naturally occurring molecules which can base pair witheach other, and allow specific amplification of bacterial genomic DNA.As an example, pyDAD and puADA may be incorporated into primers andsubstrate molecules as appropriate (Sismour et al., Nucleic AcidsResearch, 2004, Vol. 32, No. 2: 728-735).

Preferably, the homology is less than about 5%, less than about 10%,less than about 12.5%, less than about 15%, less than about 20%, lessthan about 30%, less than about 40%, 50%, 60%, 70% or 80% sequenceidentity with the corresponding nucleotide sequence from the one or morebacteria or other micro-organisms which produce the ATP or NAD-dependentligase which is to be detected in the sample. In one embodiment, thereis no sequence identity with the corresponding nucleotide sequence fromthe one or more bacteria or other micro-organisms which produce the ATPor NAD-dependent ligase which is to be detected in the sample overapproximately 10, 20, 30, 40 or 50 contiguous nucleotides. In anotherembodiment, there is less than about 10% or less than about 12.5%, 15%,20%, 30%, 40%, 50% or 60% sequence identity over approximately 10, 20,30, 40 or 50 contiguous nucleotides with the corresponding nucleotidesequence from the one or more bacteria or other micro-organisms whichproduce the ATP or NAD-dependent ligase which is to be detected in thesample.

A further step of the methods of the invention comprises, consistsessentially of or consists of incubating the sample under conditionssuitable for ATP and/or NAD-dependent ligase activity. Any suitableconditions may be employed, as would be readily determined by one ofskill in the art. For example ligation may occur at any temperaturebetween around-4 and 80° C. depending upon the ligase concerned(thermophilic bacteria may be detected using reactions incubated athigher temperatures than mesophilic bacteria for example). Preferredincubation temperatures are between around 4 and 40° C., more preferablybetween around 20 and 37° C. and most preferably at room temperature forgeneral (viable) bacterial or fungal detection. Suitable incubationtimes may be between approximately 10 minutes and 10 hours, such asbetween around 30 minutes, 1 hour or 2 hours and 5, 6, 7, 8 or 9 hours.Incubation may occur in a suitable buffer. Commercially available ligasebuffers include E. coli ligase buffer available from NEB. Suitableincubation conditions for use of a ligase are well known in the art andare recommended with commercially available ligases. A suitable cofactormay be added to the sample in order to facilitate detection of theappropriate microbial ligase. For fungal cells this may be ATP, whereasfor bacterial cells NAD may be added.

In embodiments where the sample is assessed to distinguish between thepresence of NAD-dependent ligase expressing bacteria (in particulareubacteria) and ATP-dependent ligase expressing fungi the conditions maybe altered to permit detection of the respective ligase activities. Thismay involve splitting the sample and testing for NAD-dependent ligaseactivity specifically in one portion of the sample and for ATP-dependentligase activity specifically in another, or the other, portion of thesample. The splitting may occur before or after the step of treating thesample under conditions that inhibit the activity of ATP-dependentligase from mammalian cells but which do not inhibit the activity of themicrobial ligases. In each respective sample, only the appropriatecofactor (ATP or NAD) may be added to permit any suitable ligaseactivity in that sample to be detected. The absence of the essentialcofactor should prevent the other ligase from being detected. Ifrequired, the sample portion may be depleted of any endogenous cofactorprior to testing for ligase activity. For example luciferase may beadded to a sample to deplete the sample of ATP. Suitable enzymes such asoxidoreductases may be used to deplete the sample of NAD prior to ligasedetection.

The methods of the invention may incorporate suitable controls. This maybe useful in conjunction with certain sensitive detection techniques,such as nucleic acid amplification techniques (as described herein) toensure that accurate results have been obtained. For example, thecontrols may incorporate testing a sample in which microbial (ATP and/orNAD-dependent) ligase activity is known to be present. If no ligatednucleic acid molecule is produced when the substrate is added to thissample, it is clear there is a problem for example with the reagentsused in the methods or with the detection technique. A suitable negativecontrol may be a sample in which there is known to be no ATP orNAD-dependent ligase activity. Again, a positive result/detection ofsimilar levels of product as are found in the test sample is anindication that there is a problem. A control in which no nucleic acidbased substrate molecule is added may also be employed to ensure themethods are not detecting an unrelated ligation event. All combinationsand permutations of appropriate controls are envisaged in the presentinvention. Suitable controls for use in nucleic acid amplificationreactions are employed in specific embodiments of the invention, asdescribed herein.

In preferred embodiments of the invention, the novel nucleic acidmolecule, produced according to the presence of microbial (ATP orNAD-dependent) ligase activity in the sample (as an indicator of thepresence of one or more (viable) micro-organisms, in particular fungiand/or bacteria in the sample), is detected using nucleic acidamplification techniques.

This serves to make the methods of the invention maximally sensitive.Such amplification techniques are well known in the art, and includemethods such as PCR, NASBA (Compton, 1991), 3SR (Fahy et al., 1991),Rolling circle replication, Transcription Mediated Amplification (TMA),strand displacement amplification (SDA) Clinical Chemistry 45: 777-784,1999, the DNA oligomer self-assembly processes described in U.S. Pat.No. 6,261,846 (incorporated herein by reference), ligase chain reaction(LCR) (Barringer et al., 1990), selective amplification of targetpolynucleotide sequences (U.S. Pat. No. 6,410,276), arbitrarily primedPCR (WO 90/06995), consensus sequence primed PCR (U.S. Pat. No.4,437,975), invader technology, strand displacement technology and nickdisplacement amplification (WO 2004/067726). The list above is notintended to be exhaustive. Any nucleic acid amplification technique maybe used provided the appropriate nucleic acid product is specificallyamplified.

Amplification is achieved with the use of amplification primers specificfor the sequence of the novel/ligated nucleic acid molecule which is tobe detected. In order to provide specificity for the nucleic acidmolecules primer binding sites corresponding to a suitable region of thesequence may be selected. The skilled reader will appreciate that thenucleic acid molecules may also include sequences other than primerbinding sites which are required for detection of the novel nucleic acidmolecule produced by the NAD-dependent ligase activity in the sample,for example RNA Polymerase binding sites or promoter sequences may berequired for isothermal amplification technologies, such as NASBA, 3SRand TMA.

One or more primer binding sites may bridge the ligation boundary of thesubstrate nucleic acid molecule such that an amplification product isonly generated if ligation has occurred, for example. Alternatively,primers may bind either side of the ligation boundary and directamplification across the boundary such that an amplification product isonly generated (exponentially) if the ligated nucleic acid molecule isformed. As discussed above, primers and the substrate nucleic acidmolecule(s) may be designed to avoid non-specific amplification ofbacterial genomic DNA.

Suitable primers for use in the methods of the invention comprise,consist essentially of or consist of the nucleotide sequences set forthas SEQ ID NO: 4 and 5 and SEQ ID NO: 9 and 10 and are described in moredetail in the experimental section below. These primers form a separateaspect of the invention. It is noted that variants of these sequencesmay be utilised in the present invention. In particular, additionalsequence specific flanking sequences may be added, for example toimprove binding specificity, as required. Variant sequences may have atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%nucleotide sequence identity with the nucleotide sequences of theprimers set forth in the example. The primers may incorporate syntheticnucleotide analogues as appropriate or may be RNA or PNA based forexample, or mixtures thereof. The primers may be labelled, such as withfluorescent labels and/or FRET pairs, depending upon the mode ofdetection employed. Probes may be utilised, again which may be labelled,as desired.

Thus, in certain aspects, the methods of the invention are carried outusing nucleic acid amplification techniques in order to detect the novelnucleic acid molecule produced as a direct result of the action of ATPor NAD-dependent ligase activity on the substrate nucleic acid moleculewhich indicates the presence of a bacterial cell or other NAD-dependentligase expressing micro-organism in the sample. In certain embodimentsthe technique used is selected from PCR, NASBA, 3SR, TMA, SDA and DNAoligomer self-assembly. Detection of the amplification products may beby routine methods, such as, for example, gel electrophoresis but ispreferably carried out using real-time or end-point detection methods.

A number of techniques for real-time or end-point detection of theproducts of an amplification reaction are known in the art. Theseinclude use of intercalating fluorescent dyes such as SYBR Green I(Sambrook and Russell, Molecular Cloning—A Laboratory Manual, Thirdedition), which allows the yield of amplified DNA to be estimated basedupon the amount of fluorescence produced. Many of the real-timedetection methods produce a fluorescent read-out that may becontinuously monitored; specific examples including molecular beaconsand fluorescent resonance energy transfer probes. Real-time andend-point techniques are advantageous because they keep the reaction ina “single tube”. This means there is no need for downstream analysis inorder to obtain results, leading to more rapidly obtained results.Furthermore keeping the reaction in a “single tube” environment reducesthe risk of cross contamination and allows a quantitative output fromthe methods of the invention. This may be particularly important in thecontext of the present invention where health and safety concerns may beof paramount importance (such as in detecting potential bacterialcontamination of platelet samples for example).

Real-time and end-point quantitation of PCR reactions may beaccomplished using the TaqMan® system (Applied Biosystems), see Hollandet al; Detection of specific polymerase chain reaction product byutilising the 5′-3′ exonuclease activity of Thermus aquaticus DNApolymerase; Proc. Natl. Acad. Sci. USA 88, 7276-7280 (1991), Gelmini etal. Quantitative polymerase chain reaction-based homogeneous assay withfluorogenic probes to measure C-Erb-2 oncogene amplification. Clin.Chem. 43, 752-758 (1997) and Livak et al. Towards fully automated genomewide polymorphism screening. Nat. Genet. 9, 341-342 (19995)(incorporated herein by reference). This type of probe may begenerically referred to as a hydrolytic probe. Suitablehydrolytic/Taqman probes for use in real time or end point detection arealso provided. They may comprise, consist essentially of or consist ofthe nucleotide sequence set forth as SEQ ID NO: 11. The probe issuitably labelled, for example using the labels detailed below.

In the Molecular Beacon system, see Tyagi & Kramer. Molecularbeacons—probes that fluoresce upon hybridization. Nat. Biotechnol. 14,303-308 (1996) and Tyagi et al. Multicolor molecular beacons for allelediscrimination. Nat. Biotechnol. 16, 49-53 (1998) (incorporated hereinby reference), the beacons are hairpin-shaped probes with an internallyquenched fluorophore whose fluorescence is restored when bound to itstarget. These probes may be referred to as hairpin probes.

A further real-time fluorescence based system which may be incorporatedin the methods of the invention is Zeneca's Scorpion system, seeDetection of PCR products using self-probing amplicons and fluorescenceby Whitcombe et al. Nature Biotechnology 17, 804-807 (1 Aug. 1999).Additional real-time or end-point detection techniques which are wellknown to those skilled in the art and which are commercially availableinclude Lightcycler® technology, Amplifluour® primer technology, DzyNAprimers (Todd et al., Clinical Chemistry 46:5, 625-630 (2000)), or thePlexor™ qPCR and qRT-PCR Systems.

Thus, in further aspects of the invention the products of nucleic acidamplification are detected using real-time or end point techniques. Inspecific embodiments of the invention the real-time technique consistsof using any one of hydrolytic probes (the Taqman® system), FRET probes(Lightcycler® system), hairpin primers (Amplifluour® system), hairpinprobes (the Molecular beacons system), hairpin probes incorporated intoa primer (the Scorpion® probe system), primers incorporating thecomplementary sequence of a DNAzyme and a cleavable fluorescent DNAzymesubstrate (DzYNA), Plexor qPCR and oligonucleotide blocking systems.

In certain embodiments, the reaction mixture will contain all of; thesample under test, the substrate nucleic acid molecule(s), reagents,buffers and enzymes required for amplification of the novel (ligated)nucleic acid molecule optionally in addition to the reagents required toallow real time or end-point detection of amplification products. Thusthe entire detection method for the ATP or NAD-dependent ligase (fromthe one or more bacterial cells or fungi of interest) may occur in asingle reaction, with a quantitative output, and without the need forany intermediate washing steps. Use of a “single tube” reaction isadvantageous because there is no need for downstream analysis in orderto obtain results, leading to more rapidly obtained results. Furthermorekeeping the reaction in a “single tube” environment reduces the risk ofcross contamination and allows a quantitative output from the methods ofthe invention. Also, single tube reactions are more amenable toautomation, for example in a high throughput context.

Alternatively, the methods of the invention may be carried out instep-wise fashion. Thus, in a first step it may first be necessary toprepare the sample in a form suitable for use in the method of theinvention. For example, as discussed herein, selective cell lysis orincreasing cellular permeability may be required.

The methods of the invention may also prove to have diagnostic utility,whereby an infection may be specifically and sensitively detected in theearly stages when only minimal levels of the infecting bacterial orfungal cells expressing an ATP or NAD-dependent ligase are present andit is desired to determine which type of organism is active in theinfection. Thus, the methods of the invention may be used to diagnosethe micro-organism responsible for an infection, or a disease associatedwith the presence of a micro-organism. All aspects of the invention andsteps of the method as described herein are therefore applicable to amethod of diagnosing the organism responsible for an infection, or adisease associated with the presence of a micro-organism, such as abacterial or fungal cell.

Therefore, in one specific further aspect there is provided a method ofdiagnosing the organism responsible for an infection, or a diseaseassociated with the presence of a bacterial or fungal cell, comprising,consisting essentially of or consisting of the steps of, in a sampleobtained from the subject:

(a) contacting the sample with a nucleic acid molecule which acts as asubstrate for ATP-dependent ligase activity in the sample,(b) incubating the thus contacted sample under conditions suitable forATP-dependent ligase activity; and(c) specifically determining the presence (and/or the amount) of aligated nucleic acid molecule resulting from the action of theATP-dependent ligase on the substrate nucleic acid molecule to indicatethe presence of fungi and/or bacteria causing the infection

The method may additionally comprise:

(d) contacting the sample with a nucleic acid molecule which acts as asubstrate for NAD-dependent ligase activity in the sample,(e) incubating the thus contacted sample under conditions suitable forNAD-dependent ligase activity; and(f) specifically determining the presence (and/or the amount) of aligated nucleic acid molecule resulting from the action of theNAD-dependent ligase on the substrate nucleic acid molecule to indicatethe presence of bacteria only causing the infection.

Similarly, there is provided a method of diagnosing the organismresponsible for an infection, or a disease associated with the presenceof a bacterial or fungal cell, comprising, consisting essentially of orconsisting of the steps of, in a sample obtained from the subject:

(a) treating the sample under conditions that inhibit the mammalianbackground from ATP-dependent ligase but which do not affect microbialATP and NAD-dependent ligases(b) lysing the sample to release the microbial ATP and NAD-dependentligases(c) contacting the sample with a nucleic acid molecule which acts as asubstrate for ATP-dependent ligase activity in the sample,

The method may additionally comprise:

(d) incubating the thus contacted sample under conditions suitable forATP-dependent ligase activity; and(e) specifically determining the presence (and/or the amount) of aligated nucleic acid molecule resulting from the action of theATP-dependent ligase on the substrate nucleic acid molecule to indicatethe presence of fungi and/or bacteria causing the infection(f) contacting the sample with a nucleic acid molecule which acts as asubstrate for NAD-dependent ligase activity in the sample,(g) incubating the thus contacted sample under conditions suitable forNAD-dependent ligase activity; and(h) specifically determining the presence (and/or the amount) of aligated nucleic acid molecule resulting from the action of theNAD-dependent ligase on the substrate nucleic acid molecule to indicatethe presence of bacteria only causing the infection.

In this context the “sample” will generally be a clinical sample. Thesample being used will depend on the condition that is being tested for.Typical samples which may be used, but which are not intended to limitthe invention, include whole blood, serum, plasma, platelet and urinesamples etc. taken from a patient, most preferably a human patient. Asmentioned above, the samples will contain mammalian cells. The methodsof the invention permit mammalian cell ligase activity to be removedfrom the sample prior to detection of microbial ligase activity, thusenabling the methods to have diagnostic utility.

In a preferred embodiment, the test will be an in vitro test carried outon a sample removed from a subject.

In a further embodiment, the above-described diagnostic methods mayadditionally include the step of obtaining the sample from a subject.Methods of obtaining a suitable sample from a subject are well known inthe art. Alternatively, the method may be carried out beginning with asample that has already been isolated from the patient in a separateprocedure. The diagnostic methods will most preferably be carried out ona sample from a human, but the method of the invention may havediagnostic utility for many animals.

The diagnostic methods of the invention may be used to complement anyalready available diagnostic techniques, potentially as a method ofconfirming an initial diagnosis. Alternatively, the methods may be usedas a preliminary diagnosis method in their own right, since the methodsprovide a quick and convenient means of diagnosis. Furthermore, due totheir inherent sensitivity, the diagnostic methods of the inventionrequire only a minimal sample, thus preventing unnecessary invasivesurgery. Also, a large but non-concentrated sample may also be testedeffectively according to the methods of the invention.

Thus, the methods of the invention have multiple applications beyonddetection of contaminating organisms in a sample. The descriptionprovided above with respect to the basic detection aspects of theinvention apply mutatis mutandis to the further aspects of the inventionand is not repeated for reasons of conciseness. For example, all stepsof the methods and suitable controls may be incorporated into thesemethods of the invention.

In specific embodiments the microbial, more specifically NAD-dependent,ligase is derived from a pathogenic micro-organism, in particular apathogenic bacterium.

The bacterium may be any bacterium which is capable of causing infectionor disease in a subject, preferably a human subject. In one embodiment,the bacteria comprises or consists essentially of or consists of any oneor more of Staphylococcus species, in particular Staphylococcus aureusand preferably methicillin resistant strains, Enterococcus species,Streptococcus species, Mycobacterium species, in particularMycobacterium tuberculosis, Vibrio species, in particular Vibriocholerae, Salmonella and/or Escherichia coli etc. The bacteria maycomprise, consist essentially of or consist of Clostridium species andin particular C. difficile in certain embodiments. C. difficile is themajor cause of antibiotic-associated diarrhoea and colitis, a healthcareassociated intestinal infection that mostly affects elderly patientswith other underlying diseases.

In specific embodiments the microbial, more specifically ATP-dependent,ligase is derived from pathogenic fungi. The fungi may be any fungiwhich are capable of causing infection or disease in a subject,preferably a human subject. In one embodiment, the fungus comprise orconsists essentially of or consists of any one or more of Candidaalbicans, Candida glabrata, Candida tropicalis, Candida krusei, Candidaparapsilosis, Aspergillus fumigatus, Cryptococcus neoformans,Histoplasma capsulatum and Pneumocystis jirovecii.

Also provided are test kits for performing these methods of theinvention. The test kit may be a disposable test kit in certainembodiments. Each component of the test kit may be supplied in aseparate compartment or carrier, or one or more of the components may becombined—provided that the components can be stably stored together.

Thus, the invention provides a kit for use in the methods of theinvention comprising:

(a) at least one nucleic acid molecule which acts as a substrate formicrobial ligase activity in the sample(b) means for inhibiting the activity of ATP-dependent ligase frommammalian cells which means do not inhibit the activity of the microbialligases.

All aspects and embodiments of the methods of the invention applymutatis mutandis to the kits of the invention. Thus, the means forinhibiting the activity of ATP-dependent ligase from mammalian cellswhich means do not inhibit the activity of the microbial ligases maycomprise a suitable agent to alter the pH of the sample in which thereaction takes place. In particular embodiments, the agent comprises ahigh pH solution, although it may also comprise a low pH solution. Inspecific embodiments, the means for inhibiting the activity ofATP-dependent ligase from mammalian cells which means do not inhibit theactivity of the microbial ATP-dependent ligases comprises, consistsessentially of or consists of sodium hydroxide (NaOH) or sodiumcarbonate (Na2CO3) (to raise the pH) or hydrochloric acid (HCl) orsulphuric acid (H2SO4) (to lower the pH). The agent may be present inany suitable concentration or volume as would be readily appreciated byone skilled in the art. In one specific embodiment, the NaOH is 5 mMNaOH.

Treatment with suitable means for inhibiting the activity ofATP-dependent ligase from mammalian cells, as discussed herein, mayrequire initially application of an agent to selectively permeate thecell membrane of mammalian cells. Thus, the kits of the invention mayfurther comprise, consist essentially of or consist of an agent thatpermeabilizes the mammalian cell membrane but which does not permeatethe cell wall of the micro-organism. Any suitable agent may be employed.In specific embodiments, the agent is a detergent, such as Triton X-100.

The kits may further comprising primers for specific detection of aligated nucleic acid molecule produced by microbial ligase activity inthe sample on the substrate nucleic acid molecule. Suitable primerscomprise, consist essentially of or consist of the nucleotide sequencesset forth as SEQ ID NO: 4 and 5 and SEQ ID NO: 9 and 10.

In further embodiments, the at least one nucleic acid molecule isimmobilized on a solid support or is provided together with means forimmobilizing the substrate nucleic acid molecule on said solid support.The immobilization of the substrate nucleic acid molecule on a solidsupport allows effective capture of the microbial ligase from thesample. The interaction of the immobilized substrate nucleic acidmolecule with the ligase results in the generation of a novel, ligatednucleic acid molecule. Thus, the kits of the invention may furthercomprise a solid support. The substrate may or may not be providedpre-loaded on the solid support. If it is not pre-immobilized on thesolid support, suitable reagents to allow immobilization may be providedin the kit, optionally together with suitable instructions. Reagents toallow immobilization would be well known to one of skill in the art. Anymeans of immobilization may be utilised provided that it does not havean adverse effect on the implementation of the methods of the invention,especially in terms of specificity and sensitivity of detection of themicrobial ligase from the one or more target bacterial or fungal cellsor micro-organisms.

Any suitable solid support may be included in the kits of the invention.The nature of the solid support is not critical to the performance ofthe invention provided that the substrate nucleic acid molecule may beimmobilized thereon without adversely affecting microbial ligaseactivity, including the ability of the enzyme to interact with thenucleic acid molecule. Non-limiting examples of solid supports includeany of beads, such as polystyrene beads and paramagnetic beads andderivatives thereof, affinity columns, microtitre plates etc. Where thesubstrate nucleic acid molecule is in fact two (or more) nucleic acidmolecules which are ligated together, either one or both of thesubstrate nucleic acid molecules may be immobilized on a solid support.In specific embodiments, the separate substrate nucleic acid moleculesmay be immobilized on the same support as one another. This allows themolecules to be in proximity to ensure that ligation is efficient if themicrobial (bacterial and/or fungal) ligase is present in the sampleunder test. Biotin and/or the streptavidin reagents may be incorporatedin the kits to facilitate immobilisation for example.

The kit may also comprise means to facilitate lysis or to increase thepermeability of the microbial cells in the sample, to permit microbialligase activity to be detected. The discussion of suitable means hereinapplies mutatis mutandis to the kits of the invention. In oneembodiment, the kit further comprises beads to facilitate lysis ofmicrobial cells in the sample (through use of a bead-beater technique).In specific embodiments, the beads are around 1 mm in diameter tofacilitate lysis of fungal cells. For lysis of bacterial cells, smallerbeads, of around 100 μm may be employed. Thus the kit may include beadsof a range of diameters in certain embodiments.

The kit may also incorporate reagents necessary for nucleic acidamplification. Employment of nucleic acid amplification techniquesallows sensitive detection of the presence of a novel ligated nucleicacid molecule. Suitable techniques and the necessary reagents would beimmediately apparent to one skilled in the art. Thus, the kits may inparticular incorporate suitable primers for specific detection of theligated nucleic acid molecule—as discussed in greater detail herein. Thekits may also incorporate suitable reagents for real-time detection ofamplification products.

The kits may incorporate a suitable carrier in which the reactions takeplace. Advantageously, such a carrier may comprise a multi-well plate,such as a 48 or 96 well plate for example. Such a carrier allows thedetection methods to be carried out in relatively small volumes—thusfacilitating scale up and minimising the sample volume required.

The kits will typically incorporate suitable instructions. Theseinstructions permit the methods of the invention to be carried outreliably using the kits of the invention.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 shows real time assay results for the PCR assay. The curves shownare, from left to right: 24000 cells, 2400 cells, 240 cells, 0 cells,remaining traces are buffer controls.

EXPERIMENTAL SECTION

The invention will be understood with respect to the followingnon-limiting examples:

Example 1 Blood Broth Assay for Yeast Preparation of Assay Solutions:

10x T4 DNA ligase (NEB cat. # B0202S) reaction buffer 10% Triton-X-100(Sigma cat. # T8532) 5% BSA (Sigma cat. # A7906) 1M Tris Cl pH 7.5 (fromTris HCl and Tris base, Sigma cat. # T3253, T1503) pH to 7.5 H2O (Sigmacat. # W4502) through 0.2 μm filter then autoclaved, use this H2O whererequired in assay 25 DNA (sequences from MWG) 1M NaOH (Sigma cat. #221465) DTTCeramic lysis beads (1 mm diameter) supplied by Idexx Laboratories Inc.were blocked with 1 ml 5% BSA overnight and washed with 1× reactionbuffer, 1 ml of B0202SResuspend in 1× reaction buffer, 1 ml of B0202S.DNA components were dissolved (oligonucleotides suppliedby MWG Eurofins) at a working concentration of 1 ng/μl inH2O via serial dilutions in 10 mM EDTA (sigma cat. #

E7889).

The sequences were:

(SEQ ID NO: 1) S1 ACCAAAATCCCACCACAACAGAACTCACCAACCAAACACACACACAAC AAC(SEQ ID NO: 2) S2 CCACGCTCACCTCGGCTCCCTCTTCTCTGACTCCTTCC (SEQ ID NO: 3)AS GAGGTGAGCGTGGGTTGTTGTGTGTGTGTTTCC (SEQ ID NO: 4)F CCCACCACAACAGAACTCACCAACC (SEQ ID NO: 5) R GGAAGGAGTCAGAGAAGAGGGAGCCwhere F and R refer to forward and reverse primers, S1, S2 and AS arethe 3 components of the substrate.

Assay Protocol

1. Add 10 ml blood broth (diluted 1:4) to sterile 15 ml falcon tubes2. Add 10 blocked and washed ceramic beads3. Add 0.2 ml 10% Triton, invert to mix4. Spin 4000 rpm for 20 min in bench centrifuge5. Aspirate supernatant

6. Add 1 ml H2O

7. Resuspend pellet

8. Add 9 ml H2O 9. Add 0.5 ml 5% BSA

10. Add 50 μl 1M NaOH (giving 5 mM NaOH pH12), invert to mix

11. Spin 4000 rpm for 20 min

12. Aspirate supernatant, leaving dry beads, neutralise with 10 ml 50 mMTrisCl pH 7.5, mix by vortex for 20 sec13. Spin 4000 rpm for 20 min, aspirate supernatant14. Remove remaining solution15. Resuspend beads and pelleted yeast cells in 100 μl mechanical lysismix:

5% BSA 20 μl 1% Triton-X-100 10 μl 1% Tween 20 10 μl

10×T4 DNA ligase reaction buffer 10 μlAS1318 DNA 1 ng/μl 10 μl

1M DTT 1 μl H2O 39 μl

16. Transfer to mechanical lysis tubes (Sarstedt 2 ml sterile tubes cat.#72.694.006)17. Ribolyse power 5 m/sec for 45 sec, wait 2 mins, then repeat18. Short centrifuge step (2 min)

19. Incubate 37 deg C. for 30 min 20. 2 μl to PCR Thermal CycleProgramme

50 deg 2 min95 deg 15 min 1×94 deg 10 sec72 deg 5 sec 30×

The PCR mix contained 10 μl SYBR Green 2× (Eurogentec mix cat.#RT-SN2X-03+NR), F primer 10 μM 2.25 μl, R primer 10 μM 2.25 μl, H2O 3.5μl

Results

The FIGURE shows real time assay results for the PCR assay, 10 curvesare (left to right): 24000 cells, 2400 cells, 240 cells, 0 cells,remaining traces are buffer controls.

Example 2 Demonstration of the Inactivation of Host ATP-Dependent Ligasewith NaOH

Rationale. This experiment was performed in order to demonstrate theability of alkali pH to inactivate host ATP-dependent ligase releasedfrom mammalian white blood cells.

Method For Mammalian Cells

45 10 ml of blood was diluted to 50 ml with water to lyse the red cells.White cells were collected by centrifugation.

The cells were resuspended in 50 mM hepes pH 7 and lysed by ribolysis asdescribed in step 15 of example 1 above then diluted 100-fold in water.

One aliquot of the lysed cells was treated with 5 mM NaOH pH 12 for 20min whereas another aliquot remained untreated. After treatment withNaOH the lysed cells were diluted into ligase mix and tested for ligaseactivity as described above in example 1.

For Bacteria

Cultured E. coli was diluted in water and either treated with 5 mM NaOH,5 mM NaOH and 50% (v/v) BPer (Fisher Cat. No. 78243) (to lyse thebacterial cells) or with Bper only.

After treatment for 20 min, the cells were diluted into ligase mix andtested for ligase activity as described in example 1 except that E. coliDNA ligase buffer containing NAD was used.

Results

After PCR, the cycles at which the PCR became positive were recorded(see below).

White cells+NaOH 28.3White cells−NaOH 19.5E. coli+NaOH 20.5E. coli+NaOH+BPer 15.2E. coli+BPer 15.2

Conclusion

The treatment of the white cells with NaOH reduced the signal generatedby PCR by 9 cycles compared to untreated white cells. This is due toinactivation of the host ligase by NaOH. In contrast, E. coli lysed withBPer yielded the same PCR signal whether the ligase was treated withNaOH or not. This demonstrates that the ligase present in the bacteriais much more resistant to NaOH alkali treatment. If the bacteria aretreated with NaOH only, the signal is low because the bacteria remainintact and the ligase is not released into the assay.

Example 3 Blood Broth Assay for Yeast

The purpose of this experiment is to show that yeast (Candida albicansas example) can be detected sensitively even in the presence of bloodbroth.

Preparation of Assays Solutions and Components of the Substrate were asListed Above in Example 1.

Assay Protocol

A typical assay protocol is as follows.

1. To 0.25 ml 10% (v/v) Triton X-100 in a 15 ml centrifuge tube, add 10ml blood:broth and mix. Note: If spiking with bacteria or fungi, addthem at this step.2. Incubate for 5 min on the bench then centrifuge 3-4000×g for 20 min.3. Pour off the supernatant and invert tube on a tissue to dry.4. Add 1 ml H2O and pipette to resuspend.5. Add 9 ml H2O and invert to mix. Add 1 ml 50 mM NaOH and invert to mix6. Incubate 5 min on the bench then centrifuge 3-4000×g for 20 min.7. Pour off supernatant and invert tube to dry.8. Resuspend pellet in 1 ml 50 mM Tris pH 7.5, transfer to microfugetube, spin 8,000 rpm 3 min, pipette off supernatant9. Add 50 μl Ribomix and mix to resuspend pellet.10. Transfer to a 2 ml ribolysis tube containing ribolysis beads.11. Ribolyse at power 4 for 20 sec.12. Place the tube at 37° C. for 30 min for ligation.13. Spin 8 krpm 3 min

14. Remove 2 μl to PCR. Ribomix:

5% BSA 10 μl 1% triton 5 μl 1% tween 5 μl 10 × rxn buffer 5 μl(containing ATP/NAD) DNA 0.1 pmol/μl/μl 5 μl H2O 20 μl

PCR Mix:

SYBR Stratagene mix 10 μl (# 600830) F primer 10 μM 2 μl R primer 10 μM2 μl UDGase 0.4 μl Sample 2 μl Water 3.6 μl PCR PROG 55 deg 10 min 95deg 10 min  1x 95 deg 10 sec 65 10 sec 72 10 sec 40x

DNA Sequences (all Read 5′-3′)

AS DNA: (SEQ ID NO: 6)UAG UAC UUC GUG GGU UGU UGU CUC UCG CCU UCC CAG UUCGGC CGU UGU CCG AUA UCG GCU 3′ phosphate S1: (SEQ ID NO: 7)GCC GAT ATC GGA CAA CGG CCG AAC TGG GAA GGC GAG AGA CAA CAA C S2:(SEQ ID NO: 8) 5′ phosphate CC ACG AAG TAC TAG CTG GCC GTT TGT CACCGA CGC CTA 3′ phosphate F primer (SEQ ID NO: 9)GGA CAA CGG CCG AAC TGG GAA GGC G R primer (SEQ ID NO: 10)TAG GCG TCG GTG ACA AAC GGC CAG C

Results Experiment 1.a

C. albicans in culture medium vs C. albicans in blood broth (NaOHtreated).

When C. albicans was measured using the above protocol, with an NaOHtreatment step, the results were as shown in Table 1 below:

TABLE 1 Culture medium Blood broth Numerical Numerical differencedifference Ct from Ct from Number of differ- control differ- control C.albicans Ct ence (fold) Ct ence (fold) 390 CFU/mL  24.1 3.5 11.3 24.54.6 24.3 98 CFU/mL 26.1 1.5 2.8 27.0 2.1 4.3 25 CFU/mL 25.3 2.3 4.9 27.71.4 2.6 Control 27.6 0 29.1 0

Because each Ct difference represents a two-fold increase in the signal,the figures in the “numerical difference” column are given to show theactual difference. For example, 390 CFU/mL C albicans gave an 11.3-foldincrease in signal over background or a 3.5Ct difference in culturemedium.

The results show:

1. C. albicans can be measured sensitively in blood broth.2. The background signal in blood broth is very low when the NaOHtreatment has been used.

Experiment 1b Effect of High pH Exposure on Blood DNA Ligase Signal

In blood broth that is not treated with NaOH, there is a very highsignal even after the blood cells have been removed by the Triton lysisstep (step 3 above). This appears to be due to a blood lysis residuecontaining white cells. An experiment was performed according to theabove protocol using 10 mL of sterile human blood diluted to 50 mL inculture medium and measured with and without NaOH treatment, with nofungi present. The pellet at step 6 was diluted 100 fold to keep signalswithin a reasonable range

TABLE 2 −NaOH +NaOH Blood lysis residue/100 19.5 26.7 control 29.5 29.5

In the absence of NaOH the blood signal even when diluted 100-fold was10 Ct, far higher than the level seen with small amounts of C. albicans.In the presence of NaOH this background signal is reduced to 2.8 Ct.This is a difference of 7.2 Ct or a 150-fold reduction.

Experiment 1c

Effect of High pH Exposure on C. albicans

Does high pH lyse C. albicans or is the pH effect simply because theorganism remains resistant to pH because it remains intact?

C. albicans in culture medium were exposed to varying pH for 20 minbefore being tested for ligase activity as described above but withoutthe lysis step (step 12). This was compared to a routine assay run at pH7.5 with the lysis step. In this case the high pH was created byexposure to sodium carbonate rather than sodium hydroxide.

TABLE 3 Signal change pH Ct Change in Ct (fold)  7.5 29 0 0  9.6 29.4 —— 10.2 28.9 0.1 0 11.2 26.8 2.2 4.6  7.5 (lysed) 26.8 2.2 4.6

The results show that there is no significant signal from C. albicans inthe absence of a lysis step, until pH 11.2. At this pH the yeast appearto be lysing and giving a signal as strong at that seen in lysed yeast.

If the above experiment is repeated but all the test pH samples arelysed instead, the results are as shown in table 4:

TABLE 4 Signal change pH Ct Change in Ct (fold)  7.5 26.2 3.6 12.1  9.627.2 2.6 6.1 10.2 27.1 2.7 6.5 11.2 26.5 3.3 9.8 control 29.8 0

This demonstrates that when the lysed yeast are exposed to high pH theyare still able to give a strong signal, with the signal at pH 11.2almost as high as the signal at pH 7.5. This is in direct contrast tothe results using blood.

Experiment 2

Effect of High pH on Saccharomyces cerevisiae

The experiment to test the effect of high pH was repeated using lysedand unlysed Saccharomyces cerevisiae. The lysis step in the case was toexpose the organism to YPER, a yeast lysis agent marketed by Pierce,instead of mechanical lysis.

TABLE 5 Numerical Ct Ct difference difference (fold) S. cerevisiae +YPER 27.3 3.0 8 S. cerevisiae + NaOH 28.3 2.0 4 S. cerevisiae + YPER +NaOH 28.3 2.0 4 Control 30.3 0

The experiment demonstrates that S. cerevisiae shows a good signal afterexposure to high pH even though it has been lysed.

Experiment 3

Effect of High pH on E. coli

The experiment to test the effect of high pH was repeated using lysedand unlysed E. coli. The lysis step in the case was to expose theorganism to BPER, a bacterial lysis agent marketed by Pierce, instead ofmechanical lysis.

TABLE 6 Numerical Ct Ct difference difference (fold) E. coli + BPER 24.75.1 34 E. coli + NaOH 26.6 3.2 9.2 E. coli + BPER + NaOH 23.6 6.2 74Control 29.8 0

The experiment demonstrates that E. coli shows an excellent signal afterexposure to high pH even though it has been lysed.

Experiment 4. Effect of High pH on Purified Bacterial DNA Ligase andMammalian Ligase

Recombinant E. coli DNA ligase (NEB catalogue number M0205) was exposedto pH 10.2 for 20 min and the signal compared to the unexposed enzyme.This was compared to exposure of blood ATP ligase activity after thesame period of exposure to pH 10.2.

TABLE 7 Numerical Ct Ct difference difference (fold) Bacterial DNAligase − NaOH 12.0 16 63,000 E. coli + NaOH 11.5 16.5 93,000 ControlBlood ATP ligase − NaOH 23.8 8.3 315 Blood ATP ligase + NaOH 33.4 — 0Control 32.1

The experiment shows the bacterial isolated enzyme (in the presence ofNAD, its substrate) to be extremely robust toward exposure to high pH.By contrast the mammalian ligase activity in the presence of ATP waseliminated at the same pH.

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and accompanyingfigures. Such modifications are intended to fall within the scope of theappended claims. Moreover, all embodiments described herein areconsidered to be broadly applicable and combinable with any and allother consistent embodiments, as appropriate.

Various publications are cited herein, the disclosures of which areincorporated by reference in their entireties.

1. A method of detecting a ligase expressing micro-organism in a samplecomprising a mammalian cell comprising: (a) treating the sample underhigh pH conditions that inhibit the activity of ATP-dependent ligasefrom mammalian cells but which do not inhibit the activity of themicrobial ligases, (b) contacting the sample or a portion of the samplewith a nucleic acid molecule which acts as a substrate for ligaseactivity in the sample, (c) incubating the thus contacted sample underconditions suitable for ligase activity; and (d) specificallydetermining the presence and/or the amount of a ligated nucleic acidmolecule resulting from the action of the ligase on the substratenucleic acid molecule to indicate the presence of the ligase expressingmicro-organism.
 2. The method of claim 1, wherein the ligase expressingmicro-organism comprises fungal or bacterial cells or both.
 3. Themethod of claim 1, wherein the ligase expressed by the micro-organismcomprises an ATP-dependent ligase, an NAD-dependent ligase or both. 4.The method of claim 3, wherein the presence of NAD-dependent ligaseactivity in the sample indicates the presence of bacterial cells. 5-7.(canceled)
 8. The method of claim 1, further comprising treatment of thesample with an agent that permeabilizes the mammalian cell membranesprior to or as part of treating the sample under high pH conditions thatinhibit the activity of ATP-dependent ligase from mammalian cells butwhich do not inhibit the activity of the microbial ATP-dependentligases.
 9. The method of claim 8, wherein the agent is a detergent. 10.The method of claim 1, further comprising lysis of cells in the sampleprior to or as part of treating the sample under high pH conditions thatinhibit the activity of ATP-dependent ligase from mammalian cells butwhich do not inhibit the activity of the microbial ATP-dependentligases.
 11. (canceled)
 12. The method of claim 1, wherein the high pHconditions that inhibit the activity of ATP-dependent ligase frommammalian cells but which do not inhibit the activity of the microbialligases comprise treating the sample with sodium hydroxide (NaOH) orsodium carbonate (Na₂CO₃).
 13. The method of claim 12, wherein: (i) theNaOH is around 5 mM NaOH; and/or (ii) the pH is around 12; and/or (iii)the treatment is carried out for around 20 minutes.
 14. (canceled) 15.(canceled)
 16. The method of claim 1, wherein the high pH conditionsthat inhibit the activity of ATP-dependent ligase from mammalian cellsbut which do not inhibit the activity of the microbial ligases comprisepH sufficiently high to cause lysis of the micro-organisms in thesample.
 17. The method of claim 16, wherein the pH is at least around11.
 18. The method of claim 1, further comprising diagnosing themicro-organism responsible for an infection, or a disease associatedwith the presence of a micro-organism.
 19. (canceled)
 20. (canceled) 21.The method according to claim 1, wherein the same nucleic acid moleculeis used as a substrate for both NAD-dependent ligase activity andATP-dependent ligase activity. 22-32. (canceled)
 33. A method ofdetecting fungal or bacterial cells or both in a sample comprising amammalian cell comprising: (a) treating the sample under high pHconditions that inhibit the mammalian background from ATP-dependentligase but which do not affect microbial ATP and NAD-dependent ligases(b) contacting the sample or a portion of the sample with a nucleic acidmolecule which acts as a substrate for ATP-dependent ligase activity inthe sample, (c) incubating the thus contacted sample under conditionssuitable for ATP-dependent ligase activity; and (d) specificallydetermining the presence (and/or the amount) of a ligated nucleic acidmolecule resulting from the action of the ATP-dependent ligase on thesubstrate nucleic acid molecule to indicate the presence of fungi and/orbacteria (e) contacting the sample or a portion of the sample with anucleic acid molecule which acts as a substrate for NAD-dependent ligaseactivity in the sample, (f) incubating the thus contacted sample underconditions suitable for NAD-dependent ligase activity; and (g)specifically determining the presence (and/or the amount) of a ligatednucleic acid molecule resulting from the action of the NAD-dependentligase on the substrate nucleic acid molecule to indicate the presenceof bacteria only.
 34. (canceled)
 35. A method of diagnosing the organismresponsible for an infection, or a disease associated with the presenceof a bacterial or fungal cell, comprising, consisting essentially of orconsisting of the steps of, in a sample obtained from a mammaliansubject: (a) treating the sample under high pH conditions that inhibitthe mammalian background from ATP-dependent ligase but which do notaffect microbial ATP and NAD-dependent ligases; (b) contacting thesample with a nucleic acid molecule which acts as a substrate forATP-dependent ligase activity in the sample; (c) incubating the thuscontacted sample under conditions suitable for ATP-dependent ligaseactivity; and (d) specifically determining the presence (and/or theamount) of a ligated nucleic acid molecule resulting from the action ofthe ATP-dependent ligase on the substrate nucleic acid molecule toindicate the presence of fungi and/or bacteria causing the infection;(e) contacting the sample with a nucleic acid molecule which acts as asubstrate for NAD-dependent ligase activity in the sample; (f)incubating the thus contacted sample under conditions suitable forNAD-dependent ligase activity; and (g) specifically determining thepresence (and/or the amount) of a ligated nucleic acid moleculeresulting from the action of the NAD-dependent ligase on the substratenucleic acid molecule to indicate the presence of bacteria only causingthe infection.
 36. (canceled)
 37. A method of detecting fungal cells ina sample comprising a mammalian cell comprising: (a) treating the sampleunder high pH conditions that inhibit the mammalian background signalfrom ATP-dependent ligase but which do not affect fungal ATP dependentligases (b) contacting the sample or a portion of the sample with anucleic acid molecule which acts as a substrate for ATP-dependent ligaseactivity in the sample, (c) incubating the thus contacted sample underconditions suitable for ATP-dependent ligase activity; and (d)specifically determining the presence (and/or the amount) of a ligatednucleic acid molecule resulting from the action of the ATP-dependentligase on the substrate nucleic acid molecule to indicate the presenceof fungi, wherein the absence of a ligated nucleic acid moleculeindicates the absence of fungal cells in the sample.
 38. (canceled) 39.A method of diagnosing the organism responsible for an infection, or adisease associated with the presence of a bacterial or fungal cell,comprising, consisting essentially of or consisting of the steps of, ina sample obtained from a mammalian subject: (a) treating the sampleunder high pH conditions that inhibit the mammalian background fromATP-dependent ligase but which do not affect fungal ATP-dependentligases (b) lysing the sample to release the fungal ATP-dependent ligase(c) contacting the sample with a nucleic acid molecule which acts as asubstrate for ATP-dependent ligase activity in the sample, (d)incubating the thus contacted sample under conditions suitable forATP-dependent ligase activity; and (e) specifically determining thepresence (and/or the amount) of a ligated nucleic acid moleculeresulting from the action of the ATP-dependent ligase on the substratenucleic acid molecule to indicate the presence of fungi causing theinfection, wherein the absence of a ligated nucleic acid moleculeindicates the absence of fungal cells in the sample.
 40. (canceled) 41.A method according to claim 39, wherein the mammalian ATP-dependentligase is inhibited by treating the sample with 5 mM NaOH, pH 12 for 20minutes.